PROJECT SUMMARY Lamin A is a vertebrate-specific nuclear lamina component that has been implicated in a variety of cellular functions, including structural integrity of the nucleus, sensing mechanical stress, cell signaling, and chromatin organization. Point mutations in Lamin A cause a spectrum of human degenerative disorders collectively called laminopathies including muscular dystrophies, cardiomyopathies, and a multisystem disorder known as progeria. Despite intense study, the mechanisms by which Lamin A affects cellular processes and causes such striking and tissue-specific human disease phenotypes remain unclear. The central hypothesis of this project is that Lamin A functions in part as a transcription factor, specifically as a positive modulator of enhancer function. This is a new way of thinking about Lamin A function. This hypothesis is based on strong preliminary data, showing that Lamin A associates with gene promoters and enhancers in human fibroblasts and that gains and losses of Lamin A-enhancer interactions were accompanied respectively by up-regulation and down- regulation of nearby genes. The overall objective of this proposed research is to rigorously test the hypothesis that Lamin A acts as an transcriptional activator at gene regulatory regions in the mammalian genome. The aim of the R21 phase is to identify the cell-cycle stages and cell types in which Lamin A associates with gene regulatory regions and to verify that Lamin A acts as transcription regulator. The R33 phase will probe the mechanism and function of Lamin A-enhancer associations. Specifically, this phase will focus on identifying proteins interacting with Lamin A at regulatory regions, identifying subnuclear localization of Lamin A important for the associations and function, and investigating the contribution of Lamin A-chromatin interactions to development of laminopathy-related cardiovascular disease. Altogether, the project aims to prove that Lamin A acts as a transcription activator in the mammalian genome. If proven, this entirely new mechanism of action will open new research avenues to understanding vertebrate-specific mechanisms of gene regulation and the function of Lamin A in normal physiological processes and disease. It would also provide insights to the evolution of Lamin A and the biochemical properties that allow it to act as both an intermediate filament and a factor used in gene regulation. The experiments proposed would test a hypothesis that offers a direct and logical mechanistic explanation for the molecular and physical phenotypes of laminopathies.